Project Description. The long-term objective of this project is to understand the developmental roles of the T-box family of transcription factor genes. Mutation of Tbx4 results in early lethality due to failure of the allantois to fuse with the chorion and undergo vascular remodeling. We propose thatTbx4 is upstream of the Notch signaling pathway in vasculogenesis, controlling a cell fate decision between endothelial cells and pericytes, the supporting cells of blood vessels. In addition we have discovered a role for Tbx4 in hematopoiesis, as conditional mutants lacking Tbx4 after the establishment of the chorioallantoic placenta die of anemia. We will test the hypothesis that Tbx4 is essential for the differentiation and/or maintenance of hematopoietic stem cells (HSC) in the placental labyrinth. Tbx2, Tbx3 and Tbx20 homozygous mutants all die during gestation due to distinct defects in the heart. The expression domains of these genes are partially overlapping leading to the possibility of shared functions. We have uncovered a genetic interaction between Tbx2 and Tbx3 affecting postnatal viability that is likely due to genetic interaction in heart development. We will use mutations we have available to explore T-box gene interactions in the heart. The production of a conditional allele in Tbx3 will facilitate this endeavor and allow us to further investigate the role of Tbx3 in later development. Specific Aim 1: Test the hypothesis that Tbx4 is upstream of Notch signaling in vasculogenesis in the allantois. Specific Aim 2: Test the hypothesis that Tbx4 is essential for placental hematopoiesis and that placentally- derived hematopoietic stem cells seed the fetal liver. Specific Aim 3: Explore T-box gene interactions in heart development. Specific Aim 4: Produce a conditional allele of Tbx3 to examine the role of this gene in the heart and mammary gland. Relevance: T-box genes are central to many developmental processes. The discovery of placental hematopoiesis and the placental niche for HSC in the labyrinth point to a new site for fetal hematopoiesis with an important role for Tbx4. Our proposed studies have relevance to understanding stem cell biology of the hematpoietic system. Congenital heart defects (CHD) are the most common birth defects in humans and a leading cause of death in the first year of life. T-box transcription factors are critically involved in heart development and the mutations we have available provide a unique opportunity to unravel the molecular mechanisms underlying normal heart development and to identify overlapping functional roles of different T-box genes.